Tài liệu Báo cáo khoa học: Allosteric modulation of myristate and Mn(III)heme binding to human serum albumin Optical and NMR spectroscopy characterization pptx

Allosteric modulation of myristate and Mn(III)heme
binding to human serum albumin
Optical and NMR spectroscopy characterization
Gabriella Fanali
1
, Riccardo Fesce
1
, Cristina Agrati
1
, Paolo Ascenzi
2,3
and Mauro Fasano
1
1 Dipartimento di Biologia Strutturale e Funzionale, and Centro di Neuroscienze, Universita
`
dell’Insubria, Busto Arsizio (VA), Italy
2 Dipartimento di Biologia, and Laboratorio Interdisciplinare di Microscopia Elettronica, Universita
`
‘Roma Tre’, Italy
3 Istituto Nazionale per le Malattie Infettive I.R.C.C.S. ‘Lazzaro Spallanzani’, Roma, Italy
Human serum albumin (HSA) is the most prominent
protein in plasma, but it is also found in tissues and
secretions throughout the body. HSA abundance (its
concentration being 45 mgÆmL
)1
in the serum of
human adults) contributes significantly to colloid-
osmotic blood pressure. HSA, best known for its
extraordinary ligand binding capacity, is constituted
by a single nonglycosylated all-a chain of 65 kDa con-
taining three homologous domains (labelled I, II, and

III), each composed of two (A and B) subdomains.
The three domains have different binding capacity
for a broad variety of ligands such as aminoacids
(Trp and Cys), hormones, metal ions, and bilirubin.
Moreover, HSA has a high affinity for heme and is
Keywords
allostery; fatty acid binding; heme binding;
human serum albumin; NMR relaxation
Correspondence
M. Fasano, Dipartimento di Biologia
Strutturale e Funzionale, Universita
`
dell’Insubria, Via Alberto da Giussano 12,
I-21052 Busto Arsizio (VA), Italy
Fax: +39 0331 339459
Tel: +39 0331 339450
E-mail:
Website: http://fisio.dipbsf.uninsubria.it/cns/
fasano
(Received 21 April 2005, revised 25 July
2005, accepted 26 July 2005)
doi:10.1111/j.1742-4658.2005.04883.x
Human serum albumin (HSA) is best known for its extraordinary ligand
binding capacity. HSA has a high affinity for heme and is responsible for
the transport of medium and long chain fatty acids. Here, we report myri-
state binding to the N and B conformational states of Mn(III)heme–HSA
(i.e. at pH 7.0 and 10.0, respectively) as investigated by optical absorbance
and NMR spectroscopy. At pH 7.0, Mn(III)heme binds to HSA with lower
affinity than Fe(III)heme, and displays a water molecule coordinated to the
metal. Myristate binding to a secondary site FAx, allosterically coupled to

the heme site, not only increases optical absorbance of Mn(III)heme-bound
HSA by a factor of approximately three, but also increases the Mn(III)-
heme affinity for the fatty acid binding site FA1 by 10–500-fold. Cooper-
ative binding appears to occur at FAx and accessory myristate binding
sites. The conformational changes of the Mn(III)heme–HSA tertiary struc-
ture allosterically induced by myristate are associated with a noticeable
change in both optical absorbance and NMR spectroscopic properties of
Mn(III)heme–HSA, allowing the Mn(III)-coordinated water molecule to
exchange with the solvent bulk. At pH ¼ 10.0 both myristate affinity for
FAx and allosteric modulation of FA1 are reduced, whereas cooperation
of accessory sites and FAx is almost unaffected. Moreover, Mn(III)heme
binds to HSA with higher affinity than at pH 7.0 even in the absence of
myristate, and the metal-coordinated water molecule is displaced. As a
whole, these results suggest that FA binding promotes conformational
changes reminiscent of N to B state HSA transition, and appear of general
significance for a deeper understanding of the allosteric modulation of
ligand binding properties of HSA.
Abbreviations
FA, fatty acid; HSA, human serum albumin; MSE, mean square error; NMRD, nuclear magnetic relaxation dispersion.
4672 FEBS Journal 272 (2005) 4672–4683 ª 2005 FEBS
responsible for the transport of lipophilic compounds
and drugs and of medium and long chain fatty acids;
among them, myristic acid is a stereotypic ligand to
investigate fatty acid binding and transport properties
of HSA [1–8].
Fatty acids (FAs) are required for the synthesis of
membrane lipids, hormones and second messengers,
and serve as an important source of metabolic
energy. Although the binding of fatty acids to human
and bovine serum albumin has been thoroughly inves-

tigated over many years, their binding mode and
thermodynamics are still objects of debate. By combi-
ning biochemical and biophysical approaches, a com-
mon consensus view has been reached on there being
three high-affinity fatty acid binding sites, and at least
three further low affinity sites have been envisaged.
NMR studies on tryptic and peptic fragments of
bovine serum albumin have localized two high affinity
sites in domain III and one in the N-terminal half of
the protein. Structural X-ray diffraction studies have
demonstrated that HSA is able to bind up to seven
equivalents of long chain FAs at multiple binding
sites (labelled FA1 to FA7; Fig. 1) with different
affinity. In sites FA1–5 the carboxylate moiety of
fatty acids is anchored by electrostatic ⁄ polar inter-
actions; on the contrary, sites FA6–7 do not display
a clear evidence of polar interactions that keep in
place the carboxylate head of the fatty acid, thus
suggesting that sites FA6–7 are low-affinity fatty acid
binding sites [1,6,7,9–17].
The fatty acid binding site FA1, located in subdo-
main IB (Fig. 1), acts as the heme binding site as well,
with the tetrapyrrole ring arranged in a d-shaped
cavity limited by two tyrosine residues (Tyr138 and
Tyr161) that provide p-p stacking interaction with the
porphyrin and supply a donor oxygen (from Tyr161)
for the ferric heme iron. Ferric heme is secured by the
long IA-IB connecting loop that fits into the cleft
opening. Heme propionates point toward the interface
between domains I and III and are stabilized by salt

bridges with His146 and Lys190 residues [6,8].
HSA undergoes pH- and allosteric effector-depend-
ent reversible conformational isomerization(s). Between
pH 2.7 and 4.3, HSA shows a fast (F) form, character-
ized by a dramatic increase in viscosity, much lower
solubility, and a significant loss in helical content.
Between pH 4.3 and 8, in the absence of allosteric
effectors, HSA displays the normal (N) form that is
characterized by heart-shaped structure. Between
pH 4.3 and 8, in the presence of allosteric effectors,
and at pH greater than 8, in the absence of ligands,
HSA changes conformation to the basic (B) form with
loss of a-helix and an increased affinity for some lig-
ands, such as warfarin [5,18–23].
Fatty acids are effective in allosterically regulating
ligand binding to Sudlow’s site I and to the heme cleft.
Myristate regulates HSA binding properties in a com-
plex manner, involving both competitive and allosteric
mechanisms. The structural changes associated with
FAs binding can essentially be regarded as relative
domain rearrangements to the I-II and II-III inter-
faces. This allosteric regulation is not observed for short
FAs (e.g. octanoate) that preferably bind to Sudlow’s
site II and displace the specific ligands (e.g. ibuprofen)
without inducing HSA allosteric rearrangement(s).
This indicates that the hydrophobic interactions
between the long FA polymethylenic tail and HSA
drives allosteric rearrangements. In turn, Sudlow’s site
I ligands (e.g. warfarin) displace FA7, while Sudlow’s
site II ligands (e.g. ibuprofen) displace FA3 and FA4.

Moreover, heme binding to HSA displaces FA1
[6,8,13,16,23–26].
Heme binding to HSA endows this protein with
peculiar optical absorbance and magnetic spectroscopic
properties that can be used to follow ligand- and pH-
dependent conformational transition(s) [19–22,27]. In
particular, Mn(III)heme can be used instead of Fe(III)-
heme in order to increase the strength of the dipolar
interaction with water protons when their NMR relax-
ation rate is measured [19,20]. Although an even
stronger dipolar interaction could be obtained using
Fig. 1. Ribbon representation of the heart-shaped structure of HSA
with the seven fatty acid binding sites labeled (FA1 to FA7); sites
are occupied by myristate anions rendered with red sticks. N- and
C-termini of the polypeptide chain are labeled accordingly. Atomic
coordinates are taken from [6,8,13,14]. The figure was drawn using
the
SWISS PDB viewer ( />G. Fanali et al. Myristate and Mn(III)heme binding to HSA–heme
FEBS Journal 272 (2005) 4672–4683 ª 2005 FEBS 4673
Mn(II)heme, the metal undergoes oxidation under
aerobic conditions in porphyrin complexes [28].
Heme regulates allosterically drug binding to Sud-
low’s site I. In fact, heme affinity for HSA decreases
by about one order of magnitude upon warfarin bind-
ing. Reciprocally, heme binding to HSA decreases war-
farin affinity by the same extent [19]. Fe(III)heme
allosterically inhibits ligand binding to Sudlow’s site I,
possibly by stabilizing the neutral (N) state of HSA.
Vice versa, ligand binding to Sudlow’s site I impairs
Fe(III)heme–HSA formation, possibly by stabilizing

the basic (B) state of HSA [5,18,23,29–31].
Here, we report the spectroscopic analysis of the
myristate-dependent conformational changes of the N
and B states of Mn(III)heme–HSA, by optical absorb-
ance spectroscopy and NMR spectroscopy, that show
allosteric interaction(s) between FAs and Mn(III)heme
with HSA. Interestingly, FAs increase Mn(III)heme
affinity to HSA, whereas warfarin and FA7 ligands
were reported to behave in the opposite way with
respect to ferric heme binding to HSA [19,21,31].
Additionally, the affinity of Mn(III)heme for HSA and
the spectroscopic properties of the Mn(III)heme–HSA
adduct in the presence of myristate are similar to those
of the B conformational state of HSA, suggesting that
myristate binding to one or more modulatory sites
possibly drives the N to B state HSA transition.
Results
In the absence of myristate, at pH 7.0 (i.e. HSA in the
N conformational state), Mn(III)heme binds to fatty
acid-free HSA with a dissociation constant K
H
%
2.0 · 10
)5
m (Fig. 2A). Although the binding curve
does not reach saturation and therefore the K
H
value
should be considered as a lower limit, it is worth to
note that it is two order of magnitude larger than that

measured for Fe(III)heme [32]. In the presence of
1.0 · 10
)4
m myristate, the optical absorbance spec-
trum of Mn(III)heme–HSA displays a characteristic
shoulder at 440 nm with well-defined isosbestic points
(Fig. 2B).
In the presence of myristate, the expression for
HSA-bound Mn(III)heme concentration could not be
solved analytically. Four major features are evident in
optical absorbance difference (DA) curves: (a) at low
HSA concentrations, the curves are depressed by myri-
state, indicating that myristate affinity for FA1 is
higher than that of Mn(III)heme in the absence of
myristate, and Mn(III)heme binding to FA1 is pre-
cluded by competition equilibrium (left column of
Scheme 1). (b) Maximal DA values are clearly
increased in the presence of myristate, thereby indica-
ting that binding of myristate to a modulatory site
FAx increases the signal yield of the complex. The DA
value for 1.0 · 10
)5
m Mn(III)heme–HSA–myristate
complex can be estimated about A
10
* ¼ 0.33, by nor-
malizing the value observed at 1.0 · 10
)4
m myristate
and 30 lm HSA (0.255) to full Mn(III)heme binding,

based on the molar fraction of the Mn(III)heme–HSA
adduct that gave similar spectral data at pH 10.0 (see
below). Furthermore, (c) at intermediate HSA concen-
tration the binding curves rapidly rise and appear to
Fig. 2. (A) Binding isotherms for Mn(III)heme binding to fatty acid-
free HSA and to the HSA–myristate complexes, at pH 7.0 and
25.0 °C; open triangles: no myristate; solid triangles: 5.0 · 10
)6
M
myristate; open circles: 1.0 · 10
)5
M myristate; solid circles:
2.5 · 10
)5
M myristate; crossed diamonds: 5.0 · 10
)5
M myri-
state; open diamonds: 7.5 · 10
)5
M myristate; solid diamonds:
1.0 · 10
)4
M myristate. The continuous lines were obtained by
numerical fitting of the data. Values of the dissociation equilibrium
constants obtained according to Scheme 1 are given in Table 1.
(B) UV-visible spectral changes observed for a solution of
1.0 · 10
)5
M Mn(III)heme titrated with HSA (0–3.0 · 10
)5

M) in the
presence of 1.0 · 10
)4
M myristate, at pH 7.0 and 25.0 °C. The
arrows indicate the increase of HSA concentration.
Myristate and Mn(III)heme binding to HSA–heme G. Fanali et al .
4674 FEBS Journal 272 (2005) 4672–4683 ª 2005 FEBS
reach saturation for HSA concentrations well lower
than in the absence of myristate. This indicates that
binding of myristate to the modulatory site also increa-
ses the affinity of Mn(III)heme for FA1 (Scheme 1,
central column). Finally, (d) at high HSA concentra-
tion and intermediate myristate concentrations (1.0–
5.0 · 10
)5
m) the binding curves decline, suggesting
that unbinding of myristate from FAx occurs, accord-
ing to equilibrium of the framed reaction in Scheme 1.
A kinetic model was set up to numerically fit the
optical absorbance data reported in Fig. 2A. The mini-
mal core of the model was based on the competition
between Mn(III)heme and myristate for binding to
FA1 (defined by the parameters K
H
and K
M
) and on
the allosteric modulation of FA1 properties by myri-
state binding to FAx (defined by the parameters K
M

*,
K
H
M
, and possibly K
M
M1
K
M
, if myristate binding to
FA1 is also modulated; see Experimental procedures
for explanation of the notations for the equilibrium
constants). However, binding of myristate to addi-
tional FA sites must also be considered, to take into
account the decrease in free myristate concentration at
increasing concentrations of HSA; this requires the
further set of parameters K
M
S1
to K
M
S5
. For the sake
of simplicity, these constants were bound to a fixed
affinity ratio series, with K
M
S1
as a free parameter and
K
M

Sn
¼ K
M
S1
⁄ 10
(n)1) ⁄ 2
, n ¼ 2–5; this is in general
agreement with the estimates reported in the literature
[1,9,10,33]. Two further free parameters (in addition to
K
H
, K
M
, K
H
M
, K
M
* and K
M
S1
) completed the model:
the asymptotic absorbance in the absence of myristate
(A
10
) and the absorbance of 1.0 · 10
)5
m Mn(III)-
heme–HSA–myristate complex (A
10

*). However, this
simplified model did not adequately fit the experimen-
tal data (MSE ¼ 4.9 · 10
)5
); in particular, it could not
reproduce the peak followed by partial decline
observed at intermediate myristate concentrations, par-
ticularly evident for 1.0–5.0 · 10
)4
m myristate, and in
general the right part of the curves (at high [HSA]). In
order to qualitatively reproduce this feature, positive
cooperation must be introduced between at least one
of the additional FA binding sites and FAx, so that
the Mn(III)heme–HSA–myristate adduct releases myri-
state from FAx, as free myristate concentration van-
ishes, and the optical absorbance signal declines.
Several sets of parameters gave good fits to the
experimental data, yielding almost identical curves
(MSE ¼ 3.0 ± 0.1 · 10
)5
): an example of a set of fit-
ting curves is displayed as continuous lines in Fig. 2A.
All these solutions indicate a value for K
M
S1
in the
range between 1.5 · 10
)6
and 3.0 · 10

)6
m (and thus
values of the dissociation constants for the 5 additional
FA sites ranging from 2 · 10
)6
to 2 · 10
)8
m) and sug-
gest that the affinity of myristate for FAx is modulated
by additional site no. 3 or 4, with dissociation constant
in the order of 8.0 · 10
)8
to 1.3 · 10
)7
m and a 50–200-
fold decrease in FAx affinity when the coupled site
releases myristate.
Very similar fits were obtained, whether or not the
affinity of FA1 for myristate was assumed to change
when FAx is occupied. The strength of cooperative
coupling between accessory sites and FAx could also
change over a wide extent (10–500-fold decrease in
K
M
* when additional FA site no. 3 or 4 releases myri-
state) producing equally good fits. However, the set of
estimated dissociation constants for FA1 and FAx
changed quite markedly depending on the assumptions
regarding cooperativity among FA binding sites. The
best fitting values for the parameters of the model

(Scheme 1) are reported in Table 1 for two nicely
(Myr)P(…)–(…)
K
M
*
↔ (Myr)P(Myr)–(…)
K
M
Sn
↔
↔
(Myr)P(Myr)–(Myr)
n
K
M
K
M
M
K
M
(…)P(…)–(…)
K
M
*
↔ (…)P(Myr)–(…)
K
M
Sn
↔ (…)P(Myr)–(Myr)
n

K
H
K
H
M
↔
↔
↔↔
↔
K
H
M
(Hem)P(…)–(…)
K
M
H
↔ (Hem)P(Myr)–(…)
K
M
Sn
↔ (Hem)P(Myr)–(Myr)
n
Scheme 1. Allosteric and competition equilibria involving Mn(III)heme and myristate binding to HSA. Binding sites are indicated with the
notation (FA1)P(FAx)–(FAS), where FA1 is the heme binding site, acting as myristate binding site as well, FAx is a different myristate binding
site allosterically coupled to FA1, and FAS are n secondary myristate binding sites, with different affinities, allosterically uncoupled to FA1.
P ¼ protein, HSA; Myr ¼ myristate; Hem ¼ Mn(III)heme. Values of the dissociation equilibrium constants are given in Table 1 and in the
text. The framed transition is associated with a change in the optical absorption spectrum (see text).
G. Fanali et al. Myristate and Mn(III)heme binding to HSA–heme
FEBS Journal 272 (2005) 4672–4683 ª 2005 FEBS 4675
fitting models: no change in FA1 affinity for myristate,

depending on FAx binding (K
M
M
¼ K
M
), or a similar
change in FA1 affinity for both Mn(III)heme and myr-
istate (K
M
M
⁄ K
M
¼ K
H
M
⁄ K
H
); in both cases 100-fold
decrease was assumed in K
M
* when additional FA site
no. 3 releases myristate. By inspection of the model
parameters (Table 1) it is clear that the assumptions
strongly affect the estimated affinity of FAx for myri-
state (K
M
*) and, as a consequence, the magnitude of
the allosteric modulation of FA1 (K
H
M

⁄ K
H
¼
5.4 · 10
)2
vs. 5.0 · 10
)3
). The estimate of K
M
also dif-
fers by about one order of magnitude, but the differ-
ence is smaller for the estimate of K
M
M
, i.e. FA1
affinity for myristate with occupied FAx, which pre-
sumably is the relevant dissociation constant for com-
petition between Mn(III)heme and myristate with the
latter in excess.
The same model was also applied to data obtained
at pH 10.0 (Fig. 3). The model is over-defined, and
several sets of parameters give comparable fits; the
results obtained by fixing K
M
S3
to the value observed
at pH 7.0 are displayed in Table 1. The consistent
aspects, relatively independent of the model assump-
tions, are the following: (a) FA1 affinity for Mn(III)-
heme (K

H
) increases by at least one order of
magnitude with respect to pH 7.0, but both FAx
affinity and allosteric modulation of FA1 are reduced.
(b) Cooperation of accessory sites and FAx is almost
unaffected. Finally, (c) the asymptotic absorbance of
the Mn(III)heme–HSA complex (A
10
) becomes com-
parable to that of the Mn(III)heme–HSA–(FAx +
myristate) complex (A
10
*), and the latter is not
altered by the change in pH. Again, the occurrence
of well-defined isosbestic points indicate that the
binding equilibrium occurs through only two forms,
the HSA-free and the HSA-bound Mn(III)heme
(Fig. 3B).
Table 1. Values of the thermodynamic dissociation constants (M) for myristate and Mn(III)heme binding to HSA at pH 7.0 and 10.0 (Scheme 1
and see text). Assumptions:
a
K
M
M
¼ K
M
. K
M
* · 100 for unoccupied FAS3.
b

K
M
M
⁄ K
M
¼ K
H
M
⁄ K
H
. K
M
* · 100 for unoccupied FAS3.
Constant
pH
7.0
a
10.0
a
7.0
b
10.0
b
K
H
(FA1 + Heme) 1.5 · 10
)5
1.0 · 10
)6
1.7 · 10

)5
1.1 · 10
)6
K
M
(FA1 + Myr) 3.4 · 10
)7
1.1 · 10
)6
7.2 · 10
)6
1.3 · 10
)5
K
H
M
[FA1 + Heme (FAx bound)] 8.2 · 10
)7
9.3 · 10
)8
8.5 · 10
)8
7.9 · 10
)7
K
M
M
[FA1 + Myr (FAx bound)] 3.4 · 10
)7
1.1 · 10

)6
3.7 · 10
)7
9.4 · 10
)6
K
M
*(FAx + Myr) 3.2 · 10
)7
1.2 · 10
)5
5.7 · 10
)7
2.6 · 10
)6
K
M
S3
(FAS3 + Myr) 9.2 · 10
)8
9.2 · 10
)8
1.4 · 10
)7
1.4 · 10
)7
A
10
[Asympt. DA (no Myr)] 0.011 0.027 0.012 0.027
A

10
* [Asympt. DA (+ Myr)] 0.030 0.028 0.030 0.028
Mean square error 2.9 · 10
)5
9.2 · 10
)6
2.8 · 10
)5
8.8 · 10
)6
Fig. 3. (A) Binding isotherms for Mn(III)heme binding to fatty acid-
free HSA and to the HSA–myristate complex, at pH 10.0 and
25.0 °C; open triangles: no myristate; solid diamonds: 1.0 · 10
)4
M
myristate. The continuous lines were obtained by numerical fitting
of the data. Values of the dissociation equilibrium constants
obtained according to Scheme 1 are given in Table 1. (B) UV-visible
spectral changes observed for a solution of 1.0 · 10
)5
M Mn(III)-
heme titrated with HSA (0–3.0 · 10
)5
M) in the presence of
1.0 · 10
)4
M myristate, at pH 10.0 and 25.0 °C. The arrows indicate
the increase of HSA concentration.
Myristate and Mn(III)heme binding to HSA–heme G. Fanali et al .
4676 FEBS Journal 272 (2005) 4672–4683 ª 2005 FEBS

Mn(III)heme–HSA was titrated with myristate in
order to follow the conformational transition(s) associ-
ated to the fatty acid binding. As shown in Fig. 4,
binding of myristate to Mn(III)heme HSA causes the
appearance of a shoulder at 440 nm that disappears on
increasing myristate concentration due to the displace-
ment of Mn(III)heme from FA1. Here, the equilibrium
occurs through three different forms, Mn(III)heme–
HSA in the absence of myristate, Mn(III)heme–HSA
with myristate bound to site(s) other than FA1 (spec-
trum with the shoulder at 440 nm), and free Mn(III)-
heme; therefore, no isosbestic points are observed.
A consistent behavior has been observed by measur-
ing the paramagnetic contribution of Mn(III)heme to
the solvent water proton NMR relaxation rate (Eqn 1
in Experimental procedures). Figure 5 shows the relax-
ivity of fatty acid-free Mn(III)heme–HSA observed at
10 MHz, 25.0 °C, as a function of the myristate con-
centration. The relaxation rate increases, apparently
with a varying slope, up to sevenfold molar excess of
myristate, while it starts to decrease when myristate
concentration is further increased.
An overview of the conformational changes due to
both fatty acid binding and pH may be obtained by plot-
ting
1
H-NMR relaxation rate data vs. pH for the differ-
ent Mn(III)heme ⁄ HSA ⁄ fatty acid ratios. Figure 6 shows
the pH dependence curves of the observed relaxation
rate measured at 10 MHz, where this parameter is most

affected, for Mn(III)heme–HSA and Mn(III)heme–
HSA-myristate at 1 : 1 : 3, 1 : 1 : 4.5, and 1 : 1 : 6
molar ratios. Values of pK for the three titration steps,
obtained at the different Mn(III)heme–HSA-myristate
ratios, have been determined using Eqn (2) (Experimen-
tal procedures; Table 2).
Fig. 4. (A) Absorbance change measured at 440 nm for a solution
of 1.0 · 10
)5
M Mn(III)heme–HSA as a function of myristate con-
centration. Data were obtained at pH 7.0 and 25.0 °C. (B) UV-visible
absorption spectra of a solution of 1.0 · 10
)5
M Mn(III)heme–HSA
in the absence (continuous line) and in the presence of
4.5 · 10
)5
M (dotted line) and 1.0 · 10
)4
M myristate (dashed line).
Fig. 5. Change of the relaxivity measured at 10 MHz of a
1.0 · 10
)3
M solution of Mn(III)heme–HSA as a function of myri-
state concentration. Data were obtained at pH 7.0 and 25.0 °C.
Fig. 6. Water proton relaxation rates measured at 10 MHz and
25.0 °C, as functions of pH, for fatty acid-free Mn(III)heme–HSA
(solid squares), Mn(III)heme–HSA–myristate at 1 : 1 : 3 (solid tri-
angles), 1 : 1 : 4.5 (open diamonds), and 1 : 1 : 6 molar ratios (open
circles). The continuous lines were calculated according to Eqn (2).

Results of the fitting are given in Table 2. Under all the experi-
mental conditions, the Mn(III)heme–HSA concentration was
1.0 · 10
)3
M.
G. Fanali et al. Myristate and Mn(III)heme binding to HSA–heme
FEBS Journal 272 (2005) 4672–4683 ª 2005 FEBS 4677
Contributions to relaxation differ, depending on the
conformational state of HSA and on the occupancy of
the myristate binding sites. The relaxation rate change
is highest between pH 5.5 and 8.0, where HSA is in
the native form (N state). The relaxivity of the
Mn(III)heme–HSA complex increases with myristate
concentration. It should be noticed that at pH lower
than 5.5, myristate is expected to be in the protonated
form that is not able to bind HSA [34]. Conversely,
between pH 8.3 and 11.9 (i.e. where HSA is in the B
form), the contribution of Mn(III)heme–HSA-myri-
state to paramagnetic relaxation does not differ signifi-
cantly from that of fatty acid-free Mn(III)heme–HSA.
As myristate binding appears to enhance the relaxiv-
ity of Mn(III)heme–HSA, we attempted to gain more
information from the analysis of NMRD profiles at
various myristate concentrations. Figure 7 shows
NMRD profiles of fatty acid-free Mn(III)heme–HSA
and of Mn(III)heme–HSA-myristate obtained at
1 : 1 : 3, 1 : 1 : 4.5, and 1 : 1 : 6 molar ratios at
pH 7.0. Note that NMRD profiles are significantly dif-
ferent in the high field region whereas at the low
frequency limit they are almost coincident. NMRD

profiles of Mn(III)heme–HSA as a function of myri-
state concentration were also measured at pH 10.0 in
order to check whether any change occurred for the B
state of HSA as well. As shown in Fig. 7, the NMRD
profiles of Mn(III)heme–HSA at pH 10.0 do not
appear to be affected by myristate.
Optical absorbance spectra are suggestive of different
coordination modes of Mn(III)heme in the different
conformational states of HSA [35], therefore we meas-
ured the paramagnetic contribution to the
17
O-NMR
linewidth at pH 7.0 and 10.0 as a function of myristate
concentration (Fig. 8). For paramagnetic metallopro-
teins, the width of the
17
O NMR resonance is affected
by the presence of the paramagnetic metal through the
exchange of water molecules directly coordinated to the
metal center, according to Eqn (3) (Experimental pro-
cedures) [20]. Unlike protons,
17
O nuclei are negligibly
affected by dipolar coupling with nearby unpaired
electrons, and the paramagnetic broadening of the
17
O
resonance is diagnostic of the occurrence of a direct
coordination bond between water and Mn(III) [20,36].
Table 2. pK values of pH-dependent water proton relaxation rates

measured at 10 MHz and 25.0 °C of fatty acid-free Mn(III)heme–
HSA and of Mn(III)heme–HSA-myristate. pK values were obtained
by fitting data in Fig. 6 according to Eqn (2).
Mn(III)heme ⁄ HSA ⁄ fatty
acid ratio pK
1
pK
2
pK
3
1 : 1 : 0 6.60 ± 0.04 9.40 ± 0.16 11.80 ± 0.05
1 : 1 : 3 6.30 ± 0.01 8.00 ± 0.02 12.10 ± 0.03
1 : 1 : 4.5 5.43 ± 0.01 7.32 ± 0.01 11.40 ± 0.02
1 : 1 : 6 5.40 ± 0.01 7.40 ± 0.02 11.50 ± 0.03
Fig. 7. NMRD profiles of fatty acid-free Mn(III)heme–HSA (solid
squares) and of Mn(III)heme–HSA–myristate at 1 : 1 : 3 (solid tri-
angles), 1 : 1 : 4.5 (open diamonds), and 1 : 1 : 6 molar ratios (open
circles) at pH 7.0 (A) and at pH 10.0 (B). Under all the experimental
conditions, Mn(III)heme–HSA concentration was 1.0 · 10
)3
M. Data
were obtained at 25.0 °C.
Fig. 8. Paramagnetic contribution to the linewidth of the
17
O water
resonance of 1.6 · 10
)3
M solution of Mn(III)heme–HSA as a func-
tion of myristate concentration. Solid squares: pH 7.0, HSA N state;
open circles: pH 10.0, HSA B state. Data were obtained at 25.0 °C.

Myristate and Mn(III)heme binding to HSA–heme G. Fanali et al .
4678 FEBS Journal 272 (2005) 4672–4683 ª 2005 FEBS
As shown in Fig. 8, the linewidth change is significant
(about 20 Hz) for the protein in the N state, and
becomes larger (about 40 Hz) in the presence of satur-
ating concentration of myristate. On the other hand,
this contribution is almost negligible for HSA in the B
state, and starts to increase to about 20 Hz in the pres-
ence of high myristate concentration.
Discussion
Myristate binding to HSA affects the Mn(III)heme
binding properties. The results presented here indicate
that current views – seven FA binding sites, FA1
involved in ipsosterical competition with heme binding,
FAx allosterically coupled to FA1, a scale of affinity
ratios of about half a decade among FA sites, with dis-
sociation constants in the range 10
)6
)10
)8
m, and sev-
eral possible allosteric cross interactions among FA
sites [1,3,6,9–16,33] – allow us to numerically model the
experimental results with good accuracy. In particular,
modeling indicates that binding of myristate to FAx
not only increases optical absorbance of Mn(III)heme-
bound HSA by a factor of % 3, but also increases FA1
affinity for Mn(III)heme by 10–500-fold (depending on
the assumptions about possible similar changes in affin-
ity of FA1 for myristate). This brings the value of HSA

affinity for Mn(III)heme, with myristate bound to FAx,
in the range of HSA affinity for Fe(III)heme [32]. Fur-
thermore, modeling indicates that positive cooperation
between an accessory FA site (with affinity 0.8–
1.5 · 10
)7
m for myristate) and FAx is needed to
account for the shape of the DA curves (Fig. 2A).
At pH 10.0 (i.e. where HSA is in the B state),
Mn(III)heme binds more strongly to HSA than at
pH 7.0 (i.e. where HSA is in the N state) even in the
absence of myristate, with K
H
% 10
)6
m (Fig. 3).
Moreover, in the presence of saturating concentrations
of myristate, the tendency of the curve to become sig-
moidal is much attenuated, suggesting a substantial
impairment of allosteric modulation by myristate bind-
ing to FAx. Numerical analysis of the data, using the
same models that fit the data at pH 7.0, indicate that,
independently of the model assumptions, both FAx
affinity for myristate and allosteric modulation of FA1
are reduced, whereas cooperation of accessory sites
and FAx is almost unaffected. Furthermore, the
asymptotic absorbance of the Mn(III)heme–HSA
adduct (A
10
) becomes comparable to that of the

Mn(III)heme–HSA-(FAx+myristate) complex (A
10
*),
whereas the latter is not altered by the change in pH.
Taken together, these observations strongly suggest
that the conformational changes produced by changing
the pH from 7.0 to 10.0 (i.e. shifting the HSA confor-
mation from the N to the B state) is very similar to
that induced by myristate binding to site FAx. Indeed,
the HSA affinity for Mn(III)heme and the absorbance
of Mn(III)heme–HSA increase by factors of about 10
and 3, respectively, and myristate effects become much
attenuated. Still, the same interaction(s) that at pH ¼
7.0 produces marked differences among absorbance
curves at various myristate concentrations appear to
fully account for the small reshaping of the curve pro-
duced by 1.0 · 10
)4
m myristate at pH ¼ 10.0.
Myristate binding to HSA determines conformational
changes that open the FA1 cavity allowing Mn(III)-
heme binding and consequently myristate displacement.
Actually, addition of up to three moles of long-chain
FAs is reported to enhance the binding of Sudlow’s site
I (i.e. FA7) ligands, and this behaviour is usually
explained by a cooperative effect established by FA
binding to domain III (i.e. to FA4 and FA5) [26,37–39].
On the other hand, myristate bound at the limit of
subdomain IA (i.e. to FA2) was suggested to be func-
tionally linked to Sudlow’s site I [25]. It should be

noticed that binding of more than three equivalents of
myristate decreases warfarin affinity for Sudlow’s site
I, as Fe(III)heme does [19,21,31,40]. Sudlow’s site II
(i.e. FA4) ligands do not appear to be effective in
modulating Sudlow’s site I ligands and heme binding
properties [21,26].
The marked variation in the optical absorbance
spectrum of Mn(III)heme–HSA induced by myristate
binding at pH 7.0 might be explained in terms of a
change in the coordination sphere of Mn(III) [35].
Although structural data for Mn(III)heme–HSA are
not available yet, evidence for a Mn(III)-coordinated
water molecule was gained by
17
O-NMR linewidth
measurements, that showed a transverse relaxation rate
different from Fe(III)heme–HSA, where no Fe(III)-
coordinated water molecule(s) were observed [20]. On
the other hand, both X-ray structures deposited in
PDB so far for Fe(III)heme–HSA display the Tyr161
residue as the only axial ligand for Fe(III) [6,8]. In the
absence of myristate, Mn(III)heme–HSA in the N state
has a water molecule coordinated to the metal (Fig. 8)
that could provide a source for paramagnetic relaxa-
tion of the solvent water bulk. This is at difference
with Fe(III)heme, due to the different affinity of the
metals for phenolic oxygen ligands [6,8]. Nevertheless,
this contribution is not evident from the NMRD pro-
file, which is almost superimposable to that of
Mn(III)heme–HSA in the B state. This finding could

indicate that there is a water molecule coordinated at
both pH but that its exchange is limiting the relaxivity.
Therefore, the binding of myristate seems to markedly
increase the exchange rate and induce a relaxivity
G. Fanali et al. Myristate and Mn(III)heme binding to HSA–heme
FEBS Journal 272 (2005) 4672–4683 ª 2005 FEBS 4679
enhancement, although at pH 10.0 the possible increase
in the exchange rate by myristate is incapable to
induce a significant increase of the relaxivity.
At pH 10.0 (i.e. when HSA is in the B state),
17
O-NMR linewidth measurements show no evidence of
water molecules coordinated to Mn(III)heme, as already
observed in the case of Fe(III)heme–HSA. Two hypo-
theses should be taken into consideration: either the
absence of water molecules in the coordination sphere
of the metal ion, or the presence of one water molecule
with a very slow exchange rate. Myristate binding to
HSA might increase the exchange rate, thereby produ-
cing a small broadening, but this is only observed
at pH 7.0. The structural similarity of Mn(III)heme
vs. Fe(III)heme and the structural evidence of a penta-
coordinated Fe(III) atom, with no water molecules
coordinated to it, favour the first hypothesis: in this
case, upon deprotonation at pH 10.0 the phenolic
Tyr161 oxygen becomes more nucleophylic and displa-
ces the Mn(III)-coordinated water molecule with the
consequent quenching of the paramagnetic relaxation.
Conclusions
The conformational transition(s) driven by myristate

binding to HSA may be efficiently monitored by
taking advantage of the optical and relaxometric pro-
perties of the Mn(III)heme label. Mn(III)heme
binds to FA1 in the fatty acid-free HSA with
K
H
% 2.0 · 10
)5
m; myristate not only competitively
binds to FA1, but also binds to a different site(s) and
induces conformational changes that lowers the equi-
librium constant for Mn(III)heme binding to the FA1
site by a factor of 10–500 (depending on possible
modulation of myristate binding to FA1). This con-
formational change(s) also favours the exchange of the
Mn(III)-coordinated water molecule with the solvent
bulk. At pH ¼ 10.0, Mn(III)heme binds to HSA with
higher affinity even in the absence of myristate, releas-
ing the metal-coordinated water molecule.
As a general remark, NMRD data prove a valuable
complement to X-ray crystallography to add dynamic
information to structural data, and to provide thermo-
dynamic description of the binding equilibria. As an
addition to conventional optical methods, NMRD pro-
vides a useful hint to follow environment changes that
involve the coordination sphere of the paramagnetic
metal.
Experimental procedures
All reagents were purchased from Sigma-Aldrich (St Louis,
MO, USA), were of highest purity available, and were used

without further purification. HSA was essentially fatty acid-
free according to the charcoal delipidation protocol [41–43]
and used without any further purification. Absence of signi-
ficant amounts of covalent dimers was checked by
MALDI-TOF mass spectrometry. Mn(III)heme was pre-
pared as previously reported [28]. The actual concentration
of the Mn(III)heme stock solution was checked as bis-
imidazolate complex in sodium dodecyl sulfate micelles
with an extinction coefficient of 10.3 cm
)1
Æmm
)1
(at
556 nm) [44]. Mn(III)heme–HSA was prepared by adding
the appropriate volume of 3.0 · 10
)2
m Mn(III)heme dis-
solved in 1.0 · 10
)1
m NaOH to a 1.0 · 10
)3
m HSA solu-
tion in NaCl ⁄ P
i
(1.0 · 10
)2
m phosphate buffer, 0.15 m
NaCl). The final solution of Mn(III)heme–HSA was
1.0 · 10
)3

m. Under all the experimental conditions, no free
Mn(III)heme was present in the reaction mixtures. The act-
ual concentration of the HSA stock solution was deter-
mined by using the Bradford method [45].
The sodium myristate 0.1 m solution was prepared by
adding 0.1 m fatty acid to NaOH 0.1 m. The solution was
heated to 100 °C and stirred to dissolve the fatty acid.
The sodium myristate solution was then mixed with
1.0 · 10
)3
m Mn(III)heme–HSA (fatty acid free) to achieve
the desired fatty acid to protein molar ratio. The Mn(III)-
heme–HSA-myristate complex was incubated for one hour
at room temperature with continuous stirring [6]. Sample
pH was changed by adding a few lL of 0.1 m HCl or
NaOH solutions.
Binding experiments of Mn(III)heme to HSA-myristate
and titrations of Mn(III)heme–HSA with myristate were
investigated spectrophotometrically using an optical cell
with 1.0-cm path length on a Cary 50 Bio spectrophotome-
ter (Varian Inc., Palo Alto, CA, USA). In a typical experi-
ment, a small amount of a solution of Mn(III)heme in
NaOH (about 3.0 · 10
)3
m) was diluted in the optical cell
with a solution of 1.0 · 10
)4
m sodium myristate in a sol-
vent mixture of DMSO-aqueous 0.1 m phosphate buffer
pH 7.0 to a final chromophore concentration of 1.0 · 10

)5
m.
This solution was titrated with HSA by adding small
amounts of a 1.0 · 10
)3
m protein solution in the aqueous
buffer and recording the spectrum after incubation for
a few min after each addition. Difference spectra with
respect to Mn(III)heme were taken and the binding iso-
therm was analyzed by plotting the difference of absorb-
ance between the maximum and the minimum of the
two-signed difference spectra against the protein concentra-
tion [27].
Data have been numerically analyzed using the matlab
language (The MathWorks, Natick, MA, USA) according
to Scheme 1, with the following dissociation equilibrium
constants: K
H
for Mn(III)heme binding to site FA1; K
M
for
myristate binding to site FA1 and competing ipsosterically
with Mn(III)heme; K
M
* for myristate binding to site FAx,
allosterically coupled to FA1; K
H
M
for Mn(III)heme bind-
ing to the HSA–myristate complex, with myristate bound

Myristate and Mn(III)heme binding to HSA–heme G. Fanali et al .
4680 FEBS Journal 272 (2005) 4672–4683 ª 2005 FEBS
to FAx. Accordingly, the dissociation constant for myri-
state binding to FAx with FA1 occupied by Mn(III)heme
was set to K
M
H
¼ (K
M
* · K
H
M
) ⁄ K
H
[46]. Five additional
dissociation constants K
M
S1
to K
M
S5
have been introduced
in the model to take into account subtraction of myristate
by additional binding sites no. 1 to 5 essentially uncoupled
to FA1 and ⁄ or FAx. Fatty acid binding sites (FA1 to FA7)
are numbered according to literature [6,7,14].
Water proton T
1
measurements at 10 MHz, at 25.0 °C
and at variable pH were obtained on a Stelar Spinmaster-

FFC fast field cycling relaxometer (Stelar, Mede, Italy) with
16 experiments in four scans. The reproducibility in T
1
measurements was ± 0.5%.
1
H nuclear magnetic relaxation dispersion (NMRD) pro-
files were recorded at variable concentration of myristate
by measuring water proton longitudinal relaxation rates
(R
1
obs
) at magnetic field strengths in the range from
2.4 · 10
)4
to 0.235 T (corresponding to 0.01–10 MHz pro-
ton Larmor frequencies) with the field cycling relaxometer
described above.
The R
1p
relaxivity values (i.e. paramagnetic contributions
to the solvent water longitudinal relaxation rate referenced
to a 1.0 mm concentration of paramagnetic agent) were
determined by subtracting from the observed relaxation
rate (R
1
obs
) the blank relaxation rate value (R
1
dia
) measured

for the buffer at the experimental temperature, divided by
the concentration of the paramagnetic species. For
1
H nuc-
lei, R
1p
values are mostly affected by dipolar interaction
with unpaired electrons of the paramagnetic center.
Unbound water protons relax by means of diffusion–
controlled dipolar interaction (outer sphere contribution,
R
1os
– see Eqn 1), whereas for water molecules coordinated
to the metal ion or bound to the protein in close proximity
of the paramagnetic center the dipolar interaction is modu-
lated by the reorientation of the macromolecule with
respect to the applied magnetic field. The latter term is des-
cribed by Eqn (1):
R
1p
¼ R
1os
þ
½Mq
55:56
Â
1
T
1M
þ s

M
ð1Þ
where s
M
is the exchange lifetime and q is the number of
water molecules close to the metal centre. [M] is the con-
centration of the paramagnetic metal ion, and T
1M
is the
longitudinal relaxation time of localized water protons
[20,36].
Relaxivity of Mn(III)heme–HSA solutions at 25.0 ° Cas
a function of pH was analyzed according to Eqn (2):
R
1p
¼ C
0
þ
X
i
C
i
1 þ½H
þ
=K
i
ð2Þ
where C
0
is the R

1p
value at the low pH limit, K
i
is the
thermodynamic constant of the i-th titration, and C
i
is the
R
1p
change associated to the i-th titration.
17
O-NMR linewidth measurements at 25.0 °C were recor-
ded at 7.0 T on a Bruker Avance 300 spectrometer (Bruker
Biospin, Rheinstetten, Germany), equipped with a 5 mm
inner diameter tunable broadband probehead, by using a
D
2
O external lock. Sample solutions were supplemented
with enriched H
2
17
O (Cortec Ltd, Paris, France) to an iso-
topic abundance of 2%. Experimental settings: spectral
width 6.0 kHz, 90° pulse 16 ls, acquisition time 0.47 s, 128
scans, no sample spinning [20]. Paramagnetic contributions
to the
17
O-NMR linewidth (DW) were obtained by
subtracting the width of the H
2

17
O signal in the presence
of HSA from the width of the H
2
17
O signal in the
presence of Mn(III)heme–HSA at different myristate con-
centrations. DW values are related to the transverse relaxa-
tion time of the directly coordinated water oxygen (T
O
2M
)
by Eqn (3):
DW ¼
½Mq
55:56
Â
1
T
O
2M
þ s
M
ð3Þ
where s
M
is the exchange lifetime of the metal-coordinated
water molecule, [M] is the concentration of the paramag-
netic metal ion, and q is the number of water molecules
coordinated to it. The oxygen transverse relaxation time

T
O
2M
is dominated by the electron–nucleus scalar interac-
tion, that occurs only in the presence of direct oxygen-water
coordination [36].
In all figures, error bars have been omitted for clarity as
all errors have been observed to be less than 2% of the
measured values.
Acknowledgements
This work was supported by the Italian Ministry for
Instruction, University and Research. Part of the work
has been performed at the Bioindustry Park Canavese,
Colleretto Giacosa (TO), Italy.
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